Murashige-Skoog Liquid Medium Research Articles

Indole-3-acetic acid and zinc synergistically mitigate positively charged nanoplastic-induced damage in rice

Recent research has shown that polystyrene nanoplastics (PS-NPs) can inhibit plant growth and the development of crops, such as rice. In this study, we aimed to investigate the effects of PS-NPs of different particle sizes (80 nm, 200 nm, and 2 µm) and charges (negative, neutral, and positive) on rice growth, and to explore the underlying mechanisms and potential strategies for mitigating their impacts. Two-week-old rice plants were planted in a standard ½ Murashige-Skoog liquid medium holding 50 mg/L of different particle sizes and/or charged PS-NPs for 10 days, and the liquid medium without PS-NPs was used as control. The results showed that positively charged PS-NPs (80 nm PS-NH2) had the greatest impact on plant growth and greatly reduced the dry biomass, root length, and plant height of rice by 41.04%, 46.34%, and 37.45%, respectively. The positively charged NPs with a size of 80 nm significantly decreased the zinc (Zn) and indole-3-acetic acid (IAA, auxin) contents by 29.54% and 48.00% in roots, and 31.15% and 64.30% in leaves, respectively, and down-regulated the relative expression level of rice IAA response and biosynthesis genes. Moreover, Zn and/or IAA supplements significantly alleviated the adverse effects of 80 nm PS-NH2 on rice plant growth. Exogenous Zn and/or IAA increased seedlings’ growth, decreased PS-NPs distribution, maintained redox homeostasis, and improved tetrapyrrole biosynthesis in rice treated with 80 nm PS-NH2. Our findings suggest that Zn and IAA synergistically alleviate positively charged NP-induced damage in rice.

Application of temporary immersion system RITA® for efficient biomass multiplication and production of artificial seeds for ex situ conservation of Linnaea borealis L

Linnaea borealis L., belonging to the Linnaeaceae family, in some European countries including Poland, is a protected species. Phytochemical studies indicate the presence of pharmacologically important secondary metabolites, which may indicate undiscovered biological activity and untapped therapeutic potential of this species. Harvesting the raw material from natural sites is impossible due to the protection status of this glacial relict in many countries. The aim of the current work was to develop an efficient protocol of Linnaea borealis L. var. borealis shoot multiplication via the development of axillary buds in an agitated culture and a temporary-immersion system as well as to establish the production of artificial seeds via alginate encapsulated nodes for short-time storage. The highest results of growth parameters were obtained for shoots agitated in MS (Murashige & Skoog) liquid medium enriched with cytokinin, auxin and gibberellin (18.3 ± 0.4 shoots per explant and 2185 ± 98% biomass increase, respectively). The growth of shoots in the RITA® bioreactor was highly efficient, especially when hormone supplementation in MS medium was used, the amount of medium in the culture vessel was 100 or 150 ml, and the immersion time of the shoots was 1 or 2 min. The survival and recovery rates of L. borealis artificial seeds, regardless of encapsulation matrix used, subsequently decreased with the increased storage duration up to 6 months (from 100 to 60% at 4 °C and from 100 to 54% at -18 ºC, respectively). This is the first report on twinflower shoot multiplication in a temporary-immersion system and artificial seeds production of this species.

In Vitro-Propagation of Agave tequilana Weber cv. azul in a Temporary Immersion System

In Mexico, there is a need to produce large quantities of plantlets for the establishment and replanting of blue (cv. azul) agave production areas. Most of these plots are within the origin denomination area (DOT, Spanish acronym) of the distilled product of this plant, known as tequila. The objective of this study was to develop an in vitro-propagation protocol for Agave tequilana Weber cv. azul using segmented stems in both: solid and liquid media. A disinfection and in vitro technique were developed to obtain shoots, through plantlets collected in commercial plots, which attained 100% surface-disinfection and budding rate. At the multiplication stage, the effects of 6-Benzylaminopurine (BA) (0.0, 4.4 and 13.2 μM) and kinetin (0.0, 9.4, 18.8 and 37.6 μM) were evaluated on lateral-shoot production of segmented sagittal stems. These were cultivated on Murashige & Skoog (MS) medium, with the addition of 3.0% sucrose and 8 g L−1 agar. It was observed that BA and kinetin increased the number of shoots per explant, obtaining up to 18 and 26, respectively. Furthermore, it was found that just the sagittal segmentation of explants increased axillary budding. On the other hand, segmented-stem bases were grown in MS liquid medium with 3.0% sucrose, inside a RITA® system, programmed by a 5 min immersion step with a frequency of every 4 h. The effect of Indole−3-Acetic acid (IAA) (0.57, 2.9, 5.7 μM) was evaluated, while maintaining a concentration of BA (13.2 μM). It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant. These results offer a new methodology to increase the efficiency of A. tequilana Weber cv. azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.

An Efficient Somatic Embryo Liquid Culture System for Potential Use in Large-Scale and Synchronic Production of Anthurium andraeanum Seedlings.

Anthurium andraeanum Lind. is the second most important tropical flower in the world flower market. Somatic embryogenesis and plant regeneration in Anthurium has been reported previously; however, a stable and effective method for its commercial use has not been available. In this study, an efficient somatic embryogenesis and liquid culture system for large-scale production of A. andraeanum seedlings was achieved. Building on previous research, this study investigated the main factors for proembryogenic mass (PEM) proliferation, somatic embryo (SE) development, and SE germination in Anthurium. The results showed that relatively low concentrations of plant growth regulators, mineral nutrition, and sucrose promoted PEM proliferation, SE formation, and germination in a liquid culture system. This system can be described as follows: PEMs were induced from leaf blade explants on Murashige & Skoog (MS) medium with half-strength MS macronutrients (1/2 MS) containing 2.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L-1 kinetin (KT), and 3% sucrose and were proliferated in ½ MS liquid medium containing 1.0 mg L-1 2,4-D, 0.5 mg L-1 KT, and 3% sucrose. The highest proliferation coefficients were 5.11–5.16. PEMs were then transferred to MS medium with 1/8 MS macronutrients (1/8 MS) liquid medium containing 1% sucrose to develop into globular embryos and mature embryos. Finally, the mature embryos were placed on four layers of absorbent filter paper saturated with 1/8 MS liquid medium containing 1% sucrose for germination, and an average of 60 seedlings per gram SEs was obtained. This liquid culture system can be used in large-scale and synchronic production of Anthurium seedlings.

Production of Schisandrin A and Schisandrin B from Callus and Suspension Cell Cultures of Schisandra chinensis

For establishing the fermentation system of synthetic schizandrin A and schizandrin B, culture conditions of the Schisandra chinensis callus and the suspension cell were studied in this paper. The friable calluses of Schisandra chinensis (Turcz.) Baill. were induced from hypocotyls in Murashige-Skoog (MS) solidified medium supplemented with hormone 6-benzylaminopurine (6-BA) and 2,4-Dichlorophenoxyacetic butyl acetate (2,4-D) in different concentrations, and suspension cells initiated from friable callus were cultured in MS liquid medium with various concentrations and combinations of 6-BA, 2,4-D and kinetin (KT). The optimal culture condition for callus inducement was found to be MS solidified medium with 6-BA 1.0 mg/l and 2,4-D 0.3 mg/l and the optimal condition for suspension cells was MS liquid medium with 6-BA 1.0 mg/l, 2,4-D 0.2 mg/l and KT 0.5 mg/l. UPLC/Q-TOF-MS method was used for accurate identification of schisandrin A and schisandrin B in the seeds, callus and suspension cells of S. chinensis. And HPLC analytical method was used successfully for identification and quantification of these metabolites in cultures of S. chinensis. As a result, schisandrin A was obtained 0.251 mg/g, 0.118 mg/g and 0.115 mg/g from seeds, callus and suspension cells and schisandrin B was 0.142 mg/g, 0.086 mg/g and 0.05 mg/g from seeds, callus and suspension cells, respectively. Our datas indicate that callus and suspension cells had capability as seeds of S. chinensis for schisandrin A and schisandrin B synthesizing and can be used as potential sources of these biologically active lignans.

Analysis of Secondary Metabolite Production in Somatic Embryo of Pasak Bumi (Eurycoma Longifolia Jack.)

Pasak bumi (Eurycoma longifolia Jack.) has been known as a plants that can produce secondary metabolites for medicinal purposes such as: aphrosidiac, antimalaria, dysentri, antitumor, etc. Poor seed germination of pasak bumi will affect the avaibility of plant material for drug extraction. Over exploitation of this plant will also reduce plant population in its natural habitat. In vitro culture, i.e. through somatic embryogenesis, therefore, can be used as one of an alternative method for plant regeneration as well as for in vitro metabolite production. Based on this reason, the research has been done with an objective to analyze the presence of secondary metabolite in somatic embryo of pasak bumi. Seed-derived callus was used as an explant. This callus was maintained to proliferate in MS (Murashige&Skoog, 1962) medium supplemented with 2.25mg/L 2,4-D and 2.0mg/L kinetin. A half gram of callus from proliferation medium was transferred into the MS liquid medium containing 1.0 or 2.25mg/L 2,4-D, and 2.0mg/L BAP or 2.0mg/L kinetin. Histochemical examination using Jeffrey's reagen and neutral red showed that alkaloid and terpenoid substances were presence in somatic embryo of pasakbumi. In accordance with histochemical test, GC-MS analyses showed that secondary metabolites was also synthesized by non embryogenic callus and the mixture ofembryogenic callus and somatic embryo, although the concentration in the mixture of embryogenic callus and somatic embryo was lower than those in non-embryogenic callus. Secondary metabolites, including 3-[(cyclohexyl-methyl-amino)-methyl]-3H-benzooxazole-2-one (0.06%) and 2-furancarboxaldehyde, 5-(hydroxymethyl) (43.024%) were found in embryogenic callus and somatic embryo. In addition, the mixture of embryogenic callus and somatic embryo also synthesized fatty acid and lipids (52.751%) which was higher than non-embryogenic callus (24.789%). Based on the result, the mixture of embryogenic callus and somatic embryo could produce secondary metabolites, such as alkaloid, terpenoid subtances, and phenol. The concentration of metabolites in the mixture of embryogenic callus and somatic embryo, however, was lower compare to non-embryogenic callus.

Efeito de exsudatos de cultura de células de plantas em juvenis de segundo estádio de Meloidogyne incognita

Calus foram obtidos de tomateiro (Lycopersicon esculentum), cafeeiro (Coffea arabica), alfafa (Medicago sativa), orquídea (Dendrobium nobile), mostarda (Brassica rapa), batata doce (Ipomoea batatas), fumo (Nicotiana tabacum), cenoura (Daucus carota) e Crotalaria juncea em meio sólido de Murashige & Skoog (MS) seguido do cultivo em meio líquido MS em temperatura de 25-28 ºC. Após um mês, a suspensão foi passada em membrana Millipore 0,22 µm, obtendo-se, assim, o exsudato da cultura de células de cada planta testada. Ovos ou juvenis de segundo estádio (J2) de Meloidogyne incognita foram incubados nesses exsudatos e avaliadas as percentagens de eclosão, mobilidade e mortalidade dos J2. Com exceção dos ovos incubados em exsudato de orquídea, todos os demais inibiram a eclosão quando comparados com a incubação em água (testemunha). Entretanto, nos exsudatos de L. esculentum, cafeeiro e C. juncea a inibição foi mais drástica, semelhante ao aldicarb, mas significativamente diferente e menor do que em soluções contendo ingredientes do meio MS (1-5). Todos os exsudatos reduziram a mobilidade e aumentaram a mortalidade, com maior intensidade em 24 h de exposição. Porém, maior redução na mobilidade ocorreu nos exsudatos de tomateiro e alfafa, enquanto maior mortalidade no exsudato de tomateiro, seguido pelo de mostarda.

Antioxidative capacity of extracts and constituents in Cornus capitata adventitious roots.

Radical scavenging activities of extracts and constituents in Cornus capitata adventitious root cultures were evaluated by using 1,1-diphenyl-2-pycrylhydrazyl (DPPH) and superoxide anion radicals. Inhibitory activity against peroxidation of linoleic acid was assayed by using the thiobarbituric acid (TBA) method. Ethyl acetate and aqueous fractions were prepared from adventitious roots cultured in Murashige-Skoog liquid medium with 0.1 microM Cu(2+) (0.1CuMS) or 10 microM Cu(2+) (10CuMS). The highest scavenging activities on DPPH and superoxide anion radicals were observed in the ethyl acetate fraction from 0.1CuMS. In the inhibitory activity against linoleic acid oxidation, the ethyl acetate fraction from 10CuMS was highest among the fractions tested. The ethyl acetate fraction of adventitious roots cultured in 0.1CuMS contained mainly galloylglucoses (1,2,3,6-tetragalloylglucose and 1,2,3,4,6-pentagalloylglucose). The ethyl acetate fraction of adventitious roots cultured in 10CuMS contained mainly ellagic acid derivatives [3,3'-di-O-methylellagic acid 4-(5"-acetyl)-alpha-L-arabinofuranoside and stenophyllin H1]. Aqueous fractions prepared from both media contained iridoid glycosides (dihydrocornin and cornin). Tetra- and pentagalloylglucoses showed strong inhibitory activities (61.9 and 85.2%, respectively) against linoleic acid oxidation relative to those of butylated hydroxytoluene (BHT) (91.1%) or alpha-tocopherol (49.5%) at 50 microM concentration. Although both ellagic acid derivatives had weak activities (<50%) on DPPH and superoxide anion radical scavenging, 3,3'-di-O-methylellagic acid 4-(5"-acetyl)-alpha-L-arabinofuranoside was stronger (74.7%) than alpha-tocopherol (49.5%) in inhibiting linoleic acid oxidation at 50 microM concentration. Iridoid glycosides exhibited little activity against DPPH and superoxide anion radicals or against oxidation of linoleic acid.

Estabelecimento de um protocolo regenerativo para a micropropagação do abacaxizeiro

No Laboratório de Fisiologia do Desenvolvimento e Genética Vegetal do CCA-UFSC, nos anos de 1993 a 1996, foram testadas 24 combinações de tratamentos envolvendo dois genótipos de abacaxizeiro (Ananas comosus (L.) Merr.), seis combinações dos fitorreguladores ácido naftalenoacético (ANA) e 6-benzilaminopurina (BAP), e os meios de cultura líquidos e geleificados, com o objetivo de estabelecer um protocolo regenerativo para a micropropagação do abacaxizeiro. A taxa de regeneração comportou-se de forma quadrática para a maioria das combinações testadas. O meio de cultura MS (Murashige &amp; Skoog, 1962), líquido, adicionado de ANA (2,7 µM) e BAP (4,4 µM), proporcionou uma taxa média de regeneração de 19,7 brotos/explante. A magnitude do efeito genotípico exibido pelas cultivares utilizadas foi intermediária entre o efeito das combinações dos níveis de ANA e BAP (o maior) e o efeito da constituição física do meio de cultura. A melhor combinação de tratamentos foi testada em 17 acessos coletados no Estado de Santa Catarina, demonstrando-se a eficiência do protocolo regenerativo. A taxa média de regeneração foi de 15,3 brotos/explante, dos quais 40% apresentaram altura igual ou inferior a 3 cm. Brotos enraizados ou não, com altura igual ou superior a 3 cm, apresentaram valores médios de 95,5% de sobrevivência.

Ginsenosides in hairy roots of a panax hybrid1Part 3 in the series `Ginsenoside Production, Tissue Culture and Induction of Interspecific Hybrid Ginseng (Panax ginseng×P. quinquefolium)'1

Hairy roots of an interspecific hybrid ginseng ( Panax ginseng × P. quinquefolium ), named Pgq, were established by the infection of Agrobacterium rhizogenes ATCC 15834. Growth and ginsenosides content of hairy roots cultured in various basal liquid media were measured periodically from 2 to 8 weeks. In Gamborg B5 liquid medium, the hairy roots showed best growth (5.87 g fresh weight per flask) at week 8. The highest content of ginsenoside was 2.87% as dry weight at week 8 when cultured in 1/8 Murashige–Skoog liquid medium. The ginsenoside content of Pgq hairy roots was comparable to that of Pgq root cultivated in the field. However the highest yield of ginsenosides was obtained in B5 liquid medium (3.85 mg per flask at week 8).

Expression of fission yeast cdc25 alters the frequency of lateral root formation in transgenic tobacco.

Lateral root formation was examined following the expression of a fission yeast mitotic regulator gene, cdc25, under the control of a tetracycline-inducible promoter in cultured roots of tobacco. Over expression of cdc25 in fission yeast results in premature cell division at a reduced cell size. Our aim was to examine whether cdc25 expression would affect cell size in the tobacco roots, and what effect this would have on lateral root morphogenesis. Transgene integration was confirmed by Southern blotting; it was inherited as a dominant Mendelian trait. Conditions for optimal expression, determined using plants transformed with gus under the control of the same promoter, were: addition of tetracycline (5 micrograms/ml) every 72 h, to cultured roots in Murashige-Skoog liquid medium in darkness at 27 degrees C. After the addition of tetracycline, cdc25 transcripts were detected using RT-PCR, initially after 48 h, and more strongly after 72 h. Appearance of cdc25 transcripts was followed by major changes in the roots. Compared with controls, lateral root primordia were initiated more frequently, were significantly smaller and comprised smaller cells at mitosis. However, cdc25 expression did not perturb normal development of the lateral roots. The data are consistent with cdc25 expression leading to a greater frequency of lateral root primordium formation and establishing a new threshold size for cell division in the primordia which was then maintained throughout subsequent development.

Induction of Phytochelatin. (Class III Metal loth ionein) and Incorporation of Copper in Transformed Hairy Roots of Rubia tinctorum Exposed to Cadmium

Summary Hairy roots of Rubia tinctorum L. were cultured in Murashige-Skoog liquid medium containing cadmium. (Cd) chloride. (100 µmol/L), and the induction of phytochelatin including desglycyl type. (PC) was studied using a high performance liquid chromatograph connected with an inductively coupled plasmaatomic emission spectrometer. The total Cd concentration in the hairy roots was kept almost constant from days 1 to 14 during Cd exposure, but the portion that was recovered from the supernatant fraction increased with time. The increase reflected the induction of Cd-binding PC. The PC also contained copper as a metal constituent. When the metal composition in PC induced by Cd was compared in the hairy roots and the normal root cultures of R. tinctorum , the latter showed a notably higher Cu/Cd ratio, mainly due to a lower Cd level.

Polyacetylene glucosides in hairy root cultures of Lobelia cardinalis

Hairy root cultures of Lobelia cardinalis clones Lc-A, induced by Agrobacterium rhizogenes ATCC 15834, and Lc-M, induced by A. rhizogenes MAFF 03-01724, were established. In hormone-free Murashige-Skoog liquid medium (50 ml medium per flask), both clones yielded high amounts of the polyacetylene glucosides, lobetyolin (18.7 mg per flask, Lc-M) and lobetyolinin (24.7 mg per flask, Lc-A), whose production level was the maximum in all cultures investigated.

Growth and Essential Oil Production in Shoot Culture and Regenerates of Anthemis nobilis L.

Axenic shoot culture of Anthemis nobilis L. was established from young shoots of plants cultivated in a field. The shoots grew rapidly in hormone-free Murashige-Skoog liquid medium although they proliferated on the solid medium. In the shoots cultured in Murashige-Skoog liquid medium at 25°C, GC-MS analysis revealed that geranyl isovalerate was produced in greater amounts than angelates. In the roots of plantlets obtained from shoot cultures, angelates were not detectable by GC-MS, but higher content of geranyl isovalerate were detected. The plantlets obtained by culturing on hormone-free Murashige-Skoog solid medium at 25°C in 16h/day light were transplanted to pots. At the early cultivation stage, levels of geranyl isovalerate and isobutyl angelate were the same. At the later cultivation stage (20 weeks), isobutyl angelate became the main compound, and the production of geranyl isovalerate decreased gradually.

Phenolic constituents in tissue cultures of Phyllanthus niruri

Adventitious root, hairy root, shoot and crown gall cultures of Phyllanthus niruri were established and the production of several phenolic compounds was analysed. From hairy roots cultured in 1/2 Murashige-Skoog liquid medium, six phenolic compounds; gallic acid, (−)-epicatechin, (+)-gallocatechin, (−)-epigallocatechin, (−)-epicatechin 3- O-gallate and (−)-epigallocatechin 3- O-gallate were isolated. The production of these six phenols and the related compound (+)-catechin in tissue cultures were determined by HPLC. Roots of the mother plant cultivated under hydroponic conditions contained almost the same phenolic compounds as those in root cultures. On the other hand, leaves and stems of the cultivated plant contained hydrolysable tannins such as geraniin, corilagin and galloylglucose which were similarly observed in the plant collected in Peru.

A polyacetylene gentiobioside from hairy roots of Lobelia inflata

From hairy roots of Lobelia inflata, cultured in hormone-free Murashige-Skoog liquid medium, a new polyacetylene compound lobetyolinin (9- O-β- d-gentiobiosyl-2,10-tetradecadien-4,6-diyne-8,14-diol) was isolated and its structure established on the basis of chemical and spectroscopic evidence.

Tannin production in hairy root culture of Geranium thunbergii

The hairy root culture of Geranium thunbergii was established by infecting the petioles of shoot cultured in vitro with Agrobacterium rhizogenes A4. Nine tannins and related compounds, gallic acid, ellagic acid, (+)-catechin, β-glucogallin, 1,6-di- O-, 1,2,3,6-tetra- O-, 1,2,3,4,6-penta- O-galloyl-β- d-glucoses, corilagin and geraniin were isolated from the hairy roots. Hairy roots cultured in 1/2 Murashige-Skoog liquid medium mainly produced 1,2,3,4,6-penta- O-galloyl-β- d-glucose, while in Gamborg B5 liquid medium geraniin was the main compound.

Seed and Embryo Growth in Pod Cultures of Phaseolus vulgaris and P. vulgaris × P. acutifolius

Interspecific hybrid embryos resulting from crosses between Phaseolus species generally fail to reach maturity, and embryo rescue techniques are required to recover plants. To determine if ovary (pod) culture could permit interspecific hybrid embryo development, pods of P. vulgaris L. × P. acutifolius A. Gray and P. vulgaris L. (control) were cultured upright, supported by glasswool, in modified Murashige-Skoog liquid medium. The weight of seeds and length of embryos in P. vulgaris pods increased significantly during culture. However, usually only one or occasionally two seeds located at the middle positions of the pods developed to maturity. The same pod culture procedure promoted precocious germination of early cotyledonary-stage P. vulgaris × P. acutifolius embryos without further maturation of the embryos. These findings confirm that developmental arrest of the interspecific hybrid embryos in vivo is due to intrinsic deficiencies.

Purification and Properties of Glucoamylase from Sugar Beet Cells in Suspension Culture

Glucoamylase and alpha-amylase are present in callus and suspension cultures of sugar beets (Beta vulgaris L.) as well as in mature roots. The subcellular localization of glucoamylase differed in callus and suspension-cultured cells: in callus, glucoamylase was present together with alpha-amylase in the soluble fraction of cells, but in suspension cultures, it was present predominantly in the extracellular fraction while most of the alpha-amylase activity remained in cells. Glucoamylase activity was considerably lower in callus protoplasts relative to the activities of alpha-mannosidase and alpha-galactosidase and the suspension of callus in Murashige-Skoog liquid medium or in mannitol by brief agitation resulted in the release of glucoamylase to the medium. These findings suggest that glucoamylase in callus may be present in a soluble form in the free space in the cell wall. Both mature roots and callus contained alpha-amylase and glucoamylase in the soluble fraction. Glucoamylases in the soluble fraction of callus and in the medium of suspension cultures were purified separately to homogeneity by the same four-step purification procedure, which included fractionation with ammonium sulfate, column chromatography on carboxymethyl cellulose, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The identity of the glucoamylases from the two sources was confirmed by a comparison of chromatographic behavior during purification, mobility during gel electrophoresis, M(r) (83,000 D by SDS PAGE), and enzymic and kinetic properties of the catalytic reaction, such as optimal pH and temperature, heat stability, and K(m) value for soluble starch. Glucoamylase from suspension cultures was one of the major proteins that were secreted into the medium. Dedifferentiation of leaves of young plants to callus was accompanied by induction of glucoamylase and repression of some alpha-amylases and the debranching enzyme.

Acid and Alkaline Invertases in Suspension Cultures of Sugar Beet Cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K(m) values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K(m) values for sucrose.